Production extraction purification and activity assay of novel chemical compounds from cyanobacteria and microalgae

Cyanobacteria (blue- green algae) and Microalgae have emerged as an important source of high-value products, counting important bioactive and biotechnologically significant chemicals like phycobiliprotein (PBP), carotenoids and polyunsaturated fatty acids with potential biotechnological application in food, cosmetic, pharmaceuticals and nutraceuticals industries. In the first part of this thesis, optimization of culture conditions to enhance biomass and high value added compounds (phycocyanin and fucoxanthin) of cyanobacteria and microalgae was carried out by using response surface methodology (RSM). The predicted maximum biomass and phycocyanin content of Arthrospira platensis by RSM was 1.06 g L−1 and 107 mg L−1, respectively, whereas maximum biomass and phycocyanin content of 1.32 g L−1 and 127 mg L−1 was obtained in the flask validation experiments under optimized conditions (temperature 33±2 ºC, light irradiance 44 μmol photons m−2 s−1 and agitation speed 120 rpm). Further, influence of optimized conditions (temperature 33±2 ºC, light irradiance 44 μmol photons m−2 s−1 and air flow rate of 2.5 L min−1) on growth and phycocyanin content of A. platensis in 7L Panel photobioreactor (PPBR) cultivation was investigated and it was observed that a maximum biomass yield of 2.42 g L−1 with a specific growth rate 0.202 day−1 and phycocyanin content of 228 mg L−1 was obtained in the PPBR. In case of optimization of culture conditions for fucoxanthin production by P. tricornutum, the predicted maximum fucoxanthin content by RSM was 7.9 mg g−1, whereas maximum fucoxanthin content of 10.22 mg g−1 was obtained in the flask validation experiments under optimized conditions (temperature 15°C, light irradiance 55 μmol photons m−2 s−1 and agitation speed 130 rpm). Further, influence of optimized conditions (temperature 15°C, light irradiance 55 μmol photons m−2 s−1 and air flow rate of 2.5 L min−1) on fucoxanthin content of P. tricornutum in 7L Panel photobioreactor (PPBR) cultivation was investigated and it was observed that a maximum fucoxanthin content of 44.13 mg g−1 was obtained in the PPBR. Moreover, the effect of different nitrogen sources on growth, and phycobiliprotein (PBP: PC, APC and PE) composition of three cyanobacterial strains were investigated. In the batch culture period of 12 days, Phormidium sp. and Pseudoscillatoria sp. were able to utilize all tested nitrogen sources, however, ammonium chloride was the best nitrogen source for both strains to achieve maximum growth rate µ=0.284±0.03 and µ=0.274±0.01 day–1, phycobiliprotein contents 19.38±0.09% and 19.99±0.14% of dry weight, respectively. Whereas, for Arthrospira platensis, the highest growth rate of µ=0.304±0.0 day–1 and phycobiliprotein content of 22.27±0.21% of dry weight were achieved with sodium nitrate. In the second part of this thesis, different extraction methods using various extraction buffers were evaluated to understand the efficiency of extraction methods for phycocyanin and fucoxanthin extraction from A. platensis and P. tricornutum, respectively. Among the different extraction methods evaluated in this investigation, a combined method of freeze & thaw plus ultrasoication using Na-phosphate as an extraction buffer was proved to be an effective method for phycocyanin extraction and observed a maximum C-PC yield. Furthermore, phycocyanin was purified using two-step chromatographic method and the analytical grade purity (A620/A280 ratio > 4) was attained. SDS-PAGE demonstrated the purity and presence of two bands corresponding to α and β subunits of the C-phycocyanin. Whereas, among the different extraction methods studied for fucoxanthin extraction from P. tricornutum, Ultrasound assisted extraction method (UAE) using methanol as an extraction solvent was proved to be an effective method. Furthermore, fucoxanthin was successfully purified using open silica column chromatography. Moreover, the bioactivity of crude extracts and purified phycocyanin as well as fucoxanthin was assessed against different pathogenic bacteria and observed the activity against Escherichia coli, Moraxella catarrhalis, Staphylococcus aureus and Candida albicans.